Fig. 2

Redesigned marker-free CRISPR/Cas9-mediated insertion of the gfp-ruc fusion gene cassette at the Pf47 locus. a Construct used for gfp-ruc fusion gene cassette insertion into the Pf47 locus. pCBS-Pf47 was designed to target the Pf47 ORF at its 5’ terminal-region, ~150 bp from the start codon. pARM-GFP/RUCki provides a donor fragment with the gfp-ruc fusion cassette flanked by homology arms. The positions and directions of primers P5 to P8 are indicated by small black arrows. Pf EF1α 5’, Pf elongation factor 1-alpha promoter region; PbDT 3’, Pb dihydrofolate reductase terminator; ki, knock-in. (b) PCR analysis of the parasite populations obtained after transfection. d51 and d63, days 51 and 63 after transfection, respectively. (c) Luciferase assay confirming the functional expression of the integrated ruc gene in the parasites obtained after transfection. The negative control was 3D7 WT. The luciferase assays were performed in triplicate, and the standard deviations are indicated by vertical bars. (d) Live cell fluorescence microscopy of the parasites expressing the GFP protein. The negative control was 3D7 WT